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7 General conclusions

The primary goal of this study was the evaluation of the potential of comprehensive two-dimensional gas chromatography coupled to time-of-flight spectrometry (GC×GC-TOF-MS) for the analysis of wine volatiles. GC×GC-TOF-MS was used to investigate the impact of malolactic fermentation (MLF) on the volatile composition of Pinotage wines and the evaluation of a new stir bar sorptive extraction (SBSE) phase for the analysis of wine volatiles.

In the first few chapters general background on the principles of gas chromatography with emphasis on multidimensional methods and sample preparation is provided (chapter 2).

Furthermore, the volatile composition of wine and the current knowledge of the impact of malolactic fermentation on volatile wine constituents are reviewed in chapter 3.

The main part of the research is presented in chapter 4. This chapter contains the profiling of volatile compounds in MLF fermented wines. The impact of MLF on the volatile composition of wine is not well understood and very little information is available regarding the influence of MLF on Pinotage wines in particular. In this study a set of Pinotage wines fermented with several commercial LAB starter cultures under controlled conditions was therefore used. The utilization of solid phase microextraction in combination with comprehensive two-dimensional gas chromatography coupled to time-of-flight spectrometry (HS-SPME-GC×GC-TOF-MS) provided a powerful tool for the analysis of these wines. The immense separation power obtained by orthogonal separations in GC×GC allowed the identification of 115 compounds.

Moreover, enhanced sensitivity obtained by GC×GC separation allowed the analysis of compounds present in low concentrations. The identified compounds include esters, alcohols, carbonyl compounds, acids, furanes, nitrogen-containing compounds and terpenoids. The use of GC×GC led to the identification of a much larger number of compounds compared to a one-dimensional gas chromatography and the techniques therefore clearly shows promise for the detailed investigation of wine volatiles.

Furthermore, 60 compounds were quantified relative to an internal standard. The accuracy of automated peak integration was found to be insufficient for accurate statistical analysis and manual intervention was required. Subsequent combination of quantitative data with univariate and multivariate statistical data analysis allowed the distinction between wines fermented with different starter cultures and identification of the compounds responsible for this differentiation. These results point to significant metabolic differences between the starter cultures. Some compounds which showed significant difference between control and MLF wines fermented with different starter cultures, such as some minor esters, aldehydes and ketones, were linked to malolactic fermentation for the first time.

The evaluation of the suitability of a new phase for stir bar sorptive extraction (SBSE) for the

EG-Silicone Twister^® was compared with the conventional polydimethylsiloxane (PDMS) Twister. To achieve detection of a wide range of volatile wine constituents GC×GC-TOF-MS with thermal desorption (TD) was used. Several instrumental problems were encountered in the combination of the SBSE and GC×GC, although a large number of compounds were tentatively identified. The comparison of absolute peak areas demonstrated the higher affinity of especially more polar compounds for the EG-Silicone phase. However, the thermal instability resulting in interfering degradation products hampers the usage of this phase together with mass spectrometry or other non-selective detectors.

Based on these results, the suitability of the new twister phase for the extraction of thiazole, 4-methylthiazole and 2,4-dimethylthiazole from wine was further evaluated (chapter 5).

Selective nitrogen-chemiluminescence detection (NCD) with two-dimensional heart-cutting gas chromatography was successfully used to overcome the drawbacks associated with interfering degradation products of the EG-Silicone phase. The extraction performance of the EG-Silicone Twister was compared to conventional PDMS Twister in headspace and immersion modes. The EG-Silicone Twister showed much better extraction properties for all three compounds. The most polar compound, thiazole, could not be detected when using the PDMS phase. Despite the advantages of the new stir bar phase for the extraction of the thiazoles, the developed method was not sufficiently sensitive to allow analysis at the natural levels of these compounds in wine.

In the final research chapter (chapter 6), further investigation of the MLF Pinotage wines was performed. Very little is known on the impact of MLF on the composition of volatile sulfur and nitrogen compounds. Since HS-SPME-GC×GC-TOF-MS was not suited for the analysis of these highly specific wine volatiles, two established one-dimensional GC methods using headspace injection and liquid-liquid extraction in combination with sulfur selective detection were used for the analysis of sulfur- and nitrogen-containing compounds. These methods provided quantitative data for a number of important hetero-atomic compounds in the experimental wines. Similar to the HS-SPME-GC×GC-TOF-MS results, univariate and multivariate statistical methods enabled the distinction between different starter cultures based on quantitative data for 12 compounds. These data further strengthened the assumption of significant metabolic differences between the starter cultures. This study reports for the first time changes of sulfur and nitrogen containing compounds in Pinotage wines following MLF. Furthermore, differences in the concentrations of the compounds methyl 3-(methylthio)propionate and 2-aminoacetophenone were linked to MLF for the first time.

Several general conclusions may be drawn from the results presented in this thesis. In the first instance, GC×GC was shown to be a very promising technique for the analysis of wine volatiles, since it allows the separation and identification of a large number of compounds,

including trace-level volatiles, in a single analysis. Despite these advantages, however, accurate quantification of GC×GC-TOF-MS data requires extensive manual intervention, which results in intensive data analysis. There is still a need for software capable of automated non-targeted analysis of GC×GC data for screening purposes. Future combination of GC×GC with selective detectors could open a new door for the analysis of hetero-atomic compounds in wine.

Data reported here for the analysis of Pinotage wines using GC×GC and selective methods for the analysis of sulfur- and nitrogen-containing compounds have contributed significant new information regarding the effect of malolactic fermentation on the volatile compounds in these wines. Future studies in this field should focus on the metabolism of the bacterial starter cultures and the evaluation of their impact on sensory properties of wine.

Finally, the new EG-Silicone Twister phase was found to offer a useful alternative for targeted analysis of more polar compounds such as hetero-atomic compounds in wine, especially in combination with selective detectors such as sulfur and nitrogen-chemiluminescence or flame photometric detectors.